Growing demand for alternative sources of energy can be fulfilled at least in part with a renewable supply of plant-derived biofuel oil and/or ethanol. To be a viable alternative to fossil fuels, the biofuel should provide a net energy gain in production, have environmental benefits, be economically competitive, and producible in large quantities without reducing food supplies, a current unintended byproduct of existing biofuel production.
The two predominant U.S. alternative transportation fuels, relative to fossil gasoline and diesel, are ethanol fermented from corn grain starch and biodiesel oil extracted from soybean seeds. Both corn and soybean are staple crops, on which national food supply significantly relies. Corn ethanol yields 25% more energy than the energy invested in its production, whereas biodiesel yields 93% more. Relative to the fossil fuels they displace, greenhouse gas emissions are reduced 12% by the production and combustion of ethanol and 41% by biodiesel. Biodiesel also releases fewer air pollutants per net energy gain than ethanol. These advantages of biodiesel over ethanol come from lower agricultural inputs and more efficient conversion of feedstocks to fuel. However, according to a recent estimation by Hill et al. (Proc Natl Acad Sci USA. 2006, 103(30):11206-11210), even dedicating all U.S. corn and soybean production to biofuels would meet only 12% of the gasoline demand and 6% of the demand for diesel.
High biomass plants, particularly broad leaf high biomass plants, have great biofuel potential. Plants that can yield between 100-400 tons/acre of low-cost, high-value biomass materials are particularly useful especially when there are none of the high costs, labor requirements, chemical inputs, or geographic restrictions associated with low biomass plant production.
While large number of plants have been investigated as alternative energy resources, tobacco (Nicotiana tabacum and other species from the Nicotiana genus) has been mostly overlooked. Similar to hardwood trees, tobacco will coppice or re-sprout from its stump after it has been cut. Coppicing makes multiple harvests in a year possible, enabling it to produce very high biomass tonnage. Tobacco thrives on different kinds of soil in a wide range of environments. The yield of tobacco seeds amounts to 600 kg/ha. The oil content in tobacco seed ranges between 36% and 41% by weight (Giannelos P N, Zannikos S, Stournas S, Lois E, Anastoloulos G. Industrial Crops and Products 2002, 16:1-9), indicating the existence of potent oil synthesis machinery, comparable to one of the traditional oil producers, such as soybean or rapeseed. Recent experiments indicated that tobacco seed oil can partially substitute petroleum diesel fuel at most operating conditions in terms of performance parameters and emissions without any engine modification and preheating of the blends (Gunstone F. D., Pollard M., In F. D. Gunstone, ed, Structured and Modified Lipids. Marcel Dekker, New-York, pp 155-184 (2001); Usta N., Biomass and Bioenergy 2005, 28: 77-86).
Ideally, what is needed to support national alternative transportation fuel demands is biofuels produced from low-input biomass grown on agriculturally marginal land, using high-biomass plant species that are not involved in the food supply chain.
Plant lipids in the form of vegetable oil are a major plant product with great economic importance in human nutrition as well as a renewable feedstock for various industrial products and biofuels. Growing demand for alternative sources of energy can be fulfilled with a renewable supply of plant-derived fuel oil and/or ethanol. Plants represent a significant source of biofuel vegetable oil because many species accumulate oil lipids as major storage components in seeds. The main form of vegetative storage oil in seeds, which represent, depending on the species, 15-50% of seed weight, is triacylglycerol (TAG). Despite accumulation in seeds, primary oil synthesis occurs in chloroplasts of green photosynthetic tissues, with sugars as the primary precursor for fatty acid synthesis (Durret et al., 2008). However, the primary substrate for oil synthesis are the carbohydrates generated in green photosynthetic tissues (leaves and stems) that are subsequently metabolized in chloroplasts to produce free fatty acids and acetyl-CoA units, the basic building blocks for TAG. Therefore, plant leaves are the main place of building block synthesis for TAG, and as it has been experimentally examined, the amount of TAG accumulated in oil seeds may be in part determined by the amount of fatty acid produced in plastids. (Bao X, Ohlrogge J. Plant Physiol. 1999, 120:1057-62). Final storage of TAG occurs in seeds in small spherical organelles termed oil bodies (L. Planta, 1996, 208:503-511; Wahlroos T, Soukka J, Denesyuk A, Wahlroos R, Korpela T, Kilby N J. Genesis. 2003, 35(2):125-132; Katavic V, Agrawal G K, Hajduch M, Harris S L, Thelen J J.; Proteomics. 2006, 16: 4586-4598). Only about 0.2-0.3% of leaf biomass is represented by TAG. Our recent metabolic engineering efforts [Andrianov et al., 2010] doubled the amount of extractable fatty acids from the green biomass (6% of dry weight) through of diacylglycerol acyltransferase (DGAT), a key enzyme in biosynthesis of the triacylglycerol (TAG) class of lipids [Jako C et al., 2001]. In addition, see U.S. Patent Application No. 2010/0184130 (Koprowski et al.), incorporated by reference. During the course of this study the inventors determined that doubling of lipid accumulation though overexpression of DGAT was accompanied by a decrease of the fatty acid precursor pool. In addition, the inventor's previous study with high-sugar variety of tobacco NC55 demonstrated that initial higher sugar content in plant tissue is favorable for higher accumulation of lipids. The inventors determined that the expression of rate-limiting factors, such as oil biosynthetis precursors, for example increasing the availability of sugars, a primary oil synthesis precursor, could correct the imbalance and support even greater synthesis and storage of TAG in green matter.
In a previously published study, Bornke et al (2002) have found that transgenic tobacco plant bearing isomaltulose synthase gene exhibited multiple severe phenotypic alterations: young leaves were curled and developed bleached areas during maturation, flowers were misshapen and sterile. In their experiments, isomaltulose was found in elevated concentrations in several subcellular compartments, coupled with general toxic effects (Bornke F, Hajrezaei M, Heineke D, Melzer M, Herbers K, Sonnewald U., Planta, 2002, 214, 3: 356-364.). It has also been shown that overexpession of the bacterial gene coding for sucrose isomerase, which transforms sucrose into isomaltulose, significantly increased total sugar amount in potato tubers (Boernke et al., 2002a), and doubled the total sugar content in sugarcane (Wu, Birch, 2007). However expression of this enzyme in cytosol of tobacco cells caused some morphological abnormalities in plant development (Boernke et al., 2002b).